Toxin SPDP activation

Figure 21.6 SPDP can be used to activate an antibody molecule through its available amine groups to form a sulfhydryl-reactive derivative. Toxin molecules containing disulfide-linked A-B chains may be reduced with DTT to isolate the A-chain component containing a free thiol. The SPDP-activated antibody is then mixed with the reduced A chain to effect the final conjugate by disulfide bond formation.

Figure 21.7 An intact A-B subunit toxin molecule may be activated with 2-iminothiolane with good retention of cytotoxic activity. The thiolated toxin then may be conjugated with SPDP-activated antibody to generate the immunotoxin conjugate through a disulfide bond.

Protocol for Thiolation of Antibody with SPDP and Conjugation to an SPDP-Activated Toxin... 

Purify the SPDP-activated toxin from excess reagents and reaction by-products by gel filtration using a desalting resin. For the chromatography use 0.1 M sodium phosphate,... 

Conjugation of SPDP-Activated Toxin with Thiolated Antibody... 

Immediately mix the concentrated, thiolated antibody solution from part B with the SPDP-activated toxin from part A. 

Protocol for the Conjugation of SPDP-activated Antibodies with 2-Iminothiolane-Modified Toxins... 

Mix SPDP-activated antibody with thiolated gelonin in equal mass quantities (or equal volumes if they are at the same concentration). This ratio results in about a 5-fold molar excess of toxin over the amount of antibody. 

To activate the toxin, SPDP again can be used to modify the intact A—B component. After purification of the modified toxin from excess cross-linker, the SPDP—toxin is mixed with the thiolated antibody to effect the final conjugate (Fig. 319). 

SPDP-activated antibody with the thiolated toxin effects the final conjugation (Fig. 321). 

Figure 21.5 SPDP can be used to modify both an antibody and a toxin molecule for conjugation purposes. In this case, the antibody is thiolated to contain a sulfhydryl group by modification with SPDP followed by reduction with DTT. A toxin molecule is then activated with SPDP and reacted with the thiolated antibody to effect the final conjugate through a disulfide bond.

For instance, if toxin A chain-antibody conjugates are to be prepared, the antibody can be similarly activated with SPDP, but in this case not treated with reductant. After removal of... 

Another way of utilizing SPDP is to again activate the antibody to create the pyridyl disulfide derivative, but this time thiolate the toxin component using 2-iminothiolane (Chapter 1,... 

Dissolve the toxin to be conjugated in 0.1M sodium phosphate, 0.15 MNaCl, pH 7.5, at a concentration of lOmg/ml. Some protocols use as an SPDP reaction buffer, 50mM sodium borate, 0.3 M NaCl, 0.5 percent n-butanol, pH 9.0. Both buffer systems work well for the NHS ester modification reaction, although the pH 9 buffer is at the higher end of effective derivatization with active esters, since the hydrolysis rate is dramatically increased at this level of alkalinity. 

Figure 21.8 SMPT may be used to form immunotoxin conjugates by activation of the antibody component to form a thiol-reactive derivative. Reduction of an A-B toxin molecule with DTT can facilitate subsequent isolation of the A chain containing a free thiol. Mixing the A-chain containing a sulfhydryl group with the SMPT-activated antibody causes immunotoxin formation through disulfide bond linkage. The hindered disulfide of an SMPT crosslink has been found to survive in vivo for longer periods than conjugates formed with SPDP.

For instance, if toxin A chain—antibody conjugates are to be prepared, the antibody can be similarly activated with SPDP, but in this case not treated with reductant. After removal of excess cross-linker, the activated antibody can be directly mixed with isolated A chain to create the conjugate (Fig. 320). This procedure makes use of the indigenous sulfhydryl residues produced during reductive separation of the A and B chains and therefore does not require cross-linker thiolation of one of the proteins. 

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